Review of research advances in Duchenne muscular dystrophy - 2003/4

Duchenne muscular dystrophy is a muscle wasting condition caused by a genetic error in the dystrophin gene. The error results in the absence of a protein called dystrophin, which is essential to the functioning of muscle cells. Without this protein the muscle cells eventually break down and die.

Contents:


Is there a cure?

Currently there is no cure or effective treatment to significantly reduce the breakdown of muscle cells. There are however several options to manage the symptoms associated with this condition, these can be discussed with your consultant. Scientist’s worldwide continue towards finding successful therapies.

What have been the main advances this year?

In the last year there have been several important advances, which may have potential for a therapeutic treatment for Duchenne muscular dystrophy. In the UK of particular interest are developments in the ‘molecular patch’ approach. The method involves bypassing the error in the dystrophin gene to restore some of the production of dystrophin protein. This relatively new approach has shown promising preliminary results in animal and cell models. A successful bid to the UK government has now secured £1.6 million to move this research from the laboratory to initial UK safety trials.

What does this review contain?

This review contains a summary of the main developments that have been published in Duchenne muscular dystrophy over the last year. It does not seek to include every research advance, but summarises the most relevant. Further information can be obtained from the Research Department at the Muscular Dystrophy Campaign.

What are genes?

Genes are hereditary material, coded in cells that determine how an organism will look and behave. They are located on chromosomes, found inside the nucleus of cells. The chromosomes are composed of DNA, which also encodes genes, which determine the structure of all proteins in the body. Each human has an estimated 90,000 genes.

Diagram showing DNA to chromosome to cell

Duchenne muscular dystrophy results from a mutation in the dystrophin gene. The dystrophin gene is the largest identified in the human body and is located on the X chromosome (Xp21.2). About one in 3500 boys are born with a mutation which results in absence of the dystrophin protein or only small amounts being present. In around 30% of cases the mutation arises spontaneously, the remaining cases are inherited in an X-linked, recessive fashion. This means that the disease is usually inherited from the mother. As females have two copies of the X chromosome, if one is affected by the mutation the other chromosome is able to compensate. This means that dystrophin can still be produced. In this case the individual
is unlikely to have the symptoms of the disease but is said to be a carrier. Occasionally carriers display some milder symptoms and are called manifesting carriers. If a mother carries the faulty gene, her son will have a 50% chance of inheriting the condition and her daughters will have a 50% chance of becoming carriers of the disease. If the mutation is passed to a male (one X one Y chromosome), they will develop Duchenne muscular dystrophy as they do not have the extra X chromosome to compensate for the mutation, (Further information on genetics and inheritance can be obtained from the Advice and Information Service.)

The dystrophin gene is very large and so mutations can occur anywhere along its length. The majority of the gene (around 99.4%) is said not to be essential and errors - which arise in this area are unlikely to have a significant effect on the production of dystrophin protein.

Different types of mutation may occur, the most common being:

Deletions:

Part of the gene is missing completely

Duplications:

Part of the gene is copied

Point Mutation:

One letter of the gene is changed to another letter

All types of mutations in Duchenne muscular dystrophy result in no functional dystrophin (or very small quantities) being produced.

Dystrophin is found in skeletal and cardiac muscle and is localised to the area found just below the cell membrane. Although the exact function of the dystrophin protein is not understood, it is known to be involved in strengthening the cell during muscle movement and plays a role in signalling with other key muscle proteins.

If there is no dystrophin protein present, the muscle cell becomes weaker with continuous contraction and eventually dies. Initially the body is able to produce new muscle cells to replace the cells that have died, but eventually the supply is exhausted. The cells are replaced with scar and fat tissue. Forms of dystrophin exist in the brain and other parts of the body. Their exact role is not understood, but may relate to some of the other symptoms such as learning difficulties or various other symptoms observed in people with muscular dystrophies.



Developments towards treatments and cures

There is currently no cure for Duchenne muscular dystrophy. In order to find effective treatments a range of approaches are being explored including gene therapy and cell therapy. It is likely that a combination of these approaches will eventually lead to the development of treatments.

Gene transfer

Gene transfer involves the incorporation of new DNA into the cells of an organism. In the case of Duchenne muscular dystrophy a new functional dystrophin gene would be inserted into muscle cells to compensate for the non-functional one. This can be achieved in various ways. Due to the size of the dystrophin gene, inserting the gene into muscle cells has proved extremely difficult. One of the solutions has come in the form of viral vectors. Viruses are infecting organisms; they act as a microscopic parcel, delivering DNA into cells. Viruses consist of a protein shell in which their own genes are enveloped. They attach themselves to the surface of an organism’s cell and transfer their genes into it. These viruses can be manipulated so that the majority of their own genes are removed and replaced with a reporter or therapeutic gene. Different types of viruses can be used. Most commonly used is the adeno-associated virus. These are smaller viruses which are able to transport genetic material very efficiently, however they are only able to transport very small amounts of genetic material and the dystrophin gene has to be shortened to fit inside the vector.
Many of the initial experiments carried out with viruses have used the mdx mouse as a model of the human Duchenne muscular dystrophy condition. This is a laboratory animal, which has a mutation in exon 23 of the dystrophin gene. The mouse is an extremely useful model for disease, however results cannot be completely extrapolated to man and ultimately promising results will need to be checked in humans.

Yue et al, in a study published in September 2003 in Columbia, USA (1) showed how an Adeno-associated virus (AAV) could be used to carry the micro-dystrophin gene into the heart cells of mdx mice to treat cardiomyopathy. Cardiomyopathy, where the heart is weakened due to lack of the dystrophin protein, is common in patients with Duchenne muscular dystrophy.

In the experiment, an AAV containing micro-dystrophin was injected in to the mouse's heart cavity. The results were evaluated 10 months after the injection and it was shown that extensive dystrophin expression was achieved in the heart muscle. This study shows the potential for gene transfer as a possible therapy for alleviating some of the other problems associated with Duchenne muscular dystrophy.

A study in Tokyo published in March 2004, by S Takeda (2) also used AAVs to insert a micro-dystrophin, a shortened form of the dystrophin gene, into skeletal muscle cells of mdx mice. The gene was injected into the muscle cells and expression of dystrophin was measured in intervals after injection. Again they found significant expression of dystrophin in the mouse muscles.

Another solution to delivering the dystrophin gene, is to inject naked DNA into muscle cells. This type of experiment involves using a plasmid in which the gene is incorporated. A plasmid is a small circle of DNA; the gene can be added to the circle and injected into a muscle. This method means that the whole of the information in the dystrophin gene can be used instead of a shortened version. Although there are no viral proteins it is essential to check for any immune reactions to the dystrophin protein.

A study published in March 2003 by Dr Dominic Wells at Imperial College London (3) has shown that more efficient transfer of plasmids containing the dystrophin gene can be achieved by using a technique called electrotransfer. This process increases the cell permeability to allow greater amounts of genetic material to be incorporated into the cells. The use of a chemical (hyaluronidase) has improved the process further in mdx mice. The study reported that this process resulted in highly efficient gene transfer in dystrophic muscle with limited muscle damage.

A similar investigation in Japan by Murakami et al published in Feb 2003 (4) used electroporation, another term for electrotransfer. This group based at Kawasaki Medical School showed that this method increased the uptake of plasmids in mice up to nine times than without using this technique.

If human dystrophin is introduced into the muscle cells of mice, an immune reaction occurs against the foreign gene. Concerns were expressed about immune reactions to newly expressed dystrophin in Duchenne boys who were unable to produce dystrophin naturally. A study by Dominic Wells published in February 2004 (5) explored the possibility of reactions to the dystrophin protein which may be extremely pertinent in boys where there is a total absence of dystrophin. The study in mice suggests that if the body has been exposed to parts of the dystrophin protein, (even non functional forms) this may confer tolerance to the full protein. Studies in this area are ongoing.

In September 2000, Transgene together with the AFM began a safety trial for Duchenne and Becker muscular dystrophy (6). The objective was to assess the reaction of the human body to the dystrophin gene and its protein. A “naked DNA” -(non-viral) delivery strategy was used where the full-length dystrophin gene was introduced into the arm muscle using a plasmid. The dystrophin gene, produced by Professor George Dickson's group (a Muscular Dystrophy Campaign grantee), was incorporated into the Transgene plasmid under the direction of Dr Serge Braun. Professor Fardeau and his team then injected the plasmid into arm muscles in three groups of boys with Duchenne or Becker muscular dystrophy. The results released in 2003 showed that in a proportion of patients, the gene transfer resulted in production of dystrophin in a small number of fibres around the injection site (1-10% in 6 boys). The Phase 1 trial was to evaluate only the safety of the dystrophin plasmid "pharmaceutical", and not intended to produce clinical benefit. Results showed this non-viral approach to gene therapy appears in early tests to be safe and may hold promise for future therapeutic effectiveness. The safety profile was reported as excellent with no adverse reactions. Notably no signs of immune-system responses against the newly produced dystrophin protein were observed during the time of this study. The next phase of this trail is being planned.

Studies in Pittsburgh USA published in February 2004 by Liang et al (7) used the mdx mouse and introduced naked DNA via the tail vein. By clamping the major artery and vein of the lower body, the gene was effectively forced into the muscles in both legs. Widespread restoration of dystrophin protein was observed in all muscles of both legs, demonstrating a systemic type of delivery.

Gene transfer is an important area of research, however there are still problems associated with it. These include efficient transfer of the gene to all muscle, which would be required in patients with Duchenne muscular dystrophy; there is significant effort to address this problem. There are also potential problems associated with immune reactions to viral vectors and dystrophin.


Gene modification

The aim of gene modification, is to change or repair the gene sequence for therapeutic effect. One of the biggest areas of research is Exon Skipping or the ‘molecular plaster’ approach.

Dystrophin protein is produced from the dystrophin gene. In Duchenne muscular dystrophy the gene is faulty and has a mutation, which alters the coding sequence so that it no longer makes any sense. There is a specific Q and A explaining this technique of exon skipping that can be obtained from the Advice and Information Service. Scientists realised that this mutation could be ‘skipped’ over, enabling more of the code to be read and some dystrophin protein produced. The technique uses a ‘patch’ or oligonucleotides, short pieces of RNA or DNA (short pieces of code). They can be made synthetically to match exact areas of DNA including the mutation.

Gene modification can be applied in different ways:

  • Exon skipping
  • Correcting the mutation at the level of the gene itself
  • Causing the read through of a premature stop codon on a reading frame, which causes dystrophin production to stop

Firstly exon skipping attempts to turn a Duchenne mutation into a less severe Becker form. Exon skipping does not alter the gene but works so that the error is bypassed. This means that the effects of the technique are not permanent and repeated treatments would have to be performed. The entire sequence of the dystrophin gene is now known. This means that the exons that would need to be skipped in patients can be predicted in order to restore the reading frame in individuals for which this technique would be suitable. Good results have been seen in mice and culture human Duchenne muscular dystrophy cell studies. In humans the mutations occur in a range of areas, meaning that different patches would need to be developed.

The Muscular Dystrophy Campaign had been involved in a combined bid for funds from the Government to fund research into exon skipping that will lead to the first clinical safety trials in Britain. The bid involves a consortium of scientists, the Muscular Dystrophy Campaign, Parent Project UK and the Duchenne Family Support Group (details of the project can be obtained from the Advice and Information Service - email info@muscular-dystrophy.org).The bid was successful and £1.6 million has been secured to develop this area. The Muscular Dystrophy Campaign is hopeful that this research may eventually lead to a treatment for Duchenne muscular dystrophy to reduce the severity of this condition to the milder Becker form. Human trials will determine whether this technique is effective in humans.

Exon skipping research is being carried out in various centres around the world. A group in the Netherlands lead by Judith van Deutekom (8) have previously targeted 20 different exons that would, if skipped, lead to a therapy for 75% of patients. This group has demonstrated that it is possible to skip more than one exon at a time. For example they showed that a combination of antisense oligonucleotides (AONs) caused the skipping of an entire stretch of exons from exon 45 through to 51. This means that one combination of AONs would be able to treat a variety of patients with different mutations and may provide a more uniform treatment for larger groups of patients with Duchenne muscular dystrophy.

Professor Terry Partridge and colleagues, (9) funded in part by the Muscular Dystrophy Campaign, published results in July 2003 in Nature Medicine of their work in exon skipping. Encouraging results were seen from their study in the Duchenne muscular dystrophy mouse model (mdx mouse). By delivering ‘molecular patches’ together with a molecule (called F127) to enhance the delivery of the molecular patch, they demonstrated the production of significant levels of functional dystrophin without any immune response issues even on repeated administration.

In September 2003, a group led by Dr Dominic Wells at Imperial College London published a study (10), investigating another way to enhance the delivery of oligonucleotides to the areas of the body where they are needed. A chemical known as hyaluronidase was used to enhance the uptake of oligonucleotides when using electrotransfer. The study was conducted in the mdx mouse and results showed that the expression of dystrophin was improved to a level of over 20%. The results of this study reinforce the results obtained by Professor Terry Partridge and colleagues

Secondly, scientists have attempted to repair mutations at a gene level using a technique known as Chimeraplasty. This is a method of gene therapy based on the use of a molecule known as a chimeraplast, a synthetic blend of DNA and the related RNA, which tricks the patient’s own cells to repair the mutation. The chimeraplasts match the patients own DNA except for where the mutation occurs, here the molecule has the correct sequence. They attach to the DNA and activate the repair system that normally is used to repair mistakes in DNA. The patient’s body is tricked into thinking the chimeraplast is the correct version of DNA and changes the gene to match it, hopefully repairing the mutation.

Professor Thomas Rando in the USA (11) in a study published in May 2003 showed the potential of this therapeutic method by using the mdx mouse. The team used cells from the mdx mouse as a model system and showed that chimeraplasts can induce exon skipping by altering sequences at the gene level. This can reduce a Duchenne mutation to a less severe Becker form.

A recent study in Japan published in spring 2004 by Dr Matsuo (12) showed that by altering the chemistry of oligonucleotides, significant improvements could be achieved in terms of efficiency of exon skipping and reduction in toxic effects. The modified oligonucleotide used was 40 times more effective in skipping exon 19 and promoting the expression of dystrophin in model systems. New technology like this may allow the development of more efficient exon skipping drugs which are less toxic making a therapy more possible.

The third way of modifying genes involves the use of certain drugs. It has been found that a group of antibiotics called aminoglycosides can cause the protein translation machinery to ignore premature stop codons that halt the production of the dystrophin protein. About 5-15% of boys with Duchenne have this type of mutation which may respond to drugs such as Gentamicin.

Initial studies showed that in mice, full length dystrophin was produced following treatment using Gentamicin (13), however toxic effects were recorded, including stunted growth, it is also known to be toxic in humans.

A clinical trial in Italy published in 2003 (14) investigated the ability of Gentamicin to allow read through of stop codons in humans. Four patients with Duchenne muscular dystrophy having stop codons were selected who were still ambulant or had been in a wheelchair for less than four months. They received Gentamicin sulfate in two six-day cycles over a period of seven weeks. Dystrophin expression was monitored using muscle biopsies and it was found that three out of four of the patients showed some level of dystrophin expression. One patient who had a slightly different mutation showed no expression at all.

Other groups failed to reproduce these findings in humans (15). The reason for this may be explained by Gentamicin existing as five slightly different structures. These five compounds have different efficiencies and may explain the variety of results, if a different structure of Gentamicin was used each time.

The toxicity of Gentamicin has lead to the search for compounds with similar properties but with fewer side effects. An investigation in Tokyo published in Nov 2003 by Matsuda et al (16) showed that other forms of aminoglycoside antibiotics such as Negamicin could also promote read through of stop codons. The study found that Negamicin had similar effects to Gentamicin in mdx mice but was less toxic.

More recently a new chemical known as PTC124 (17) is being developed by a company in America. It is claimed that this new compound has been successfully tested in animal models of Duchenne muscular dystrophy. This compound is said to be far less toxic than Gentamicin and can also be administered orally. Initial Clinical trials with this new compound in normal volunteers are taking place this year.





Cell therapy

The study of stem cells shows how an organism is able to develop from a single cell and how healthy cells replace damaged ones in adult organisms.

Stem cells are unspecialised cells without a predetermined function. They are special because they have the ability to turn into different types of tissue or cell depending on their location. They can also be persuded to turn into specific cells in the laboratory, such as muscle cells. These unique properties have lead to the development of ‘Cell Therapy’ as a possible treatment for Duchenne muscular dystrophy.

Diagram of cell therapy on stem cells

Stem cells could be used as a possible way of regenerating tissues that have been affected by disease by introducing new, healthy cells.

Scientists usually work on two types of cell; (1) adult stem cells (or pluripotent cells), these can develop into some kinds of cells. An example is the bone marrow stem cell which develop into different types of blood cells. They typically develop into the types of tissue in which they reside. It was previously thought that adult stem cells could not be used for therapy since they were thought to be tissue specific, that is to say their function is already decided and they have passed the point at which they can change into other types of cell. Studies in mice have shown that this is not the case and they can be manipulated and persuaded to turn into different types of cells in the laboratory (18). This makes the use of adult stem cells for cell-based therapies much more interesting and a considerable amount of research is being carried out in this area.

(2) Embryonic stem cells (or totipotent cells.) These, as the name suggests are derived form embryos. They have the ability to develop into all kinds of body cells. Embryonic stem cells are usually taken from eggs that are fertilized in vitro, or in a test tube, usually in fertilization clinics. These are then donated to research with the consent of donors. When embryonic cells are grown in certain conditions they remain unspecialized, but if they are allowed to clump together they begin to differentiate or change into lots of different types of cells such as muscle and nerve cells (this is what the embryo develops from). Scientists can control the way the cells develop by manipulating the conditions they are grown in and may be able to use the resulting cells to treat certain diseases such as Parkinson’s disease, diabetes and Duchenne muscular dystrophy. The use of embryonic stem cells often has many ethical implications.

There are similarities between the two types and both have advantages and disadvantages when used for research. One of the biggest advantages of adult stem cells is the possibility of using the patient’s own stem cells to produce new cells to replace their own damaged ones. This means that the problems with immune responses could be avoided which may occur if embryonic cells were introduced from a donor. However the patient’s stem cells would need to be modified so that a working copy of the dystrophin gene could be added.

One of the main problems with stem cell therapy is making sure the cells reach as many muscles of the body as possible. An investigation in Italy published in July 2003 by Torrente et al (19) showed how the body’s circulation could be used to deliver cells around the body. The group used muscle derived stem cells (MDSCs) a rare kind of stem cell found in adult tissue that can be isolated in the laboratory and allowed to multiply. They found that when they were injected into mdx mice they attached themselves to dystrophic muscle cells. They showed that these cells had a ‘homing’ ability due to special properties of the cell. It is hoped that the discovery of these important properties and how they work will aid stem cell research in the future.

As well as the standard types of stem cell, scientists have identified different types of cells, which may have stem cell properties. A study by Louis Kunkel in America, published in March 2004 (20) identified a new type of adult stem cell known as side population cells (SP cells). Studies showed that these cells were particularly good at producing muscle tissue. The cells were injected into the muscle of mice and were found to move to the cell membrane of muscle fibres, going on to produce new muscle cells. This study was particularly interesting as the researchers showed that the process was more effective when the cells were introduced into dystrophic muscle. The study suggested that these particular cells were activated by a dystrophic environment. The researchers then used a virus vector to insert a dystrophin gene into the SP cells. These modified cells were injected into the tail veins of mdx mice. The cells migrated round the bodies and were attracted to areas where the muscle was damaged and helped repair the fibres. Dystrophin protein was detected in all the treated mdx mice but at low levels (less than 1%). This would be insufficient for a therapeutic effect at this stage. The study was important as the cell, when injected into the blood supply migrated to many muscle areas (not cardiac muscle). This raises the potential of identifying similar SP cells in Duchenne muscular dystrophy individuals, modifying these to carry dystrophin protein, then re-introducing them. So far the SP cells have only been identified in the mouse.

More research in Italy published in July 2003 by Dr Giulio Cossu (21) identified another type of stem cell known as mesoangioblasts. These stem cells have the unique ability to cross from blood vessels into muscle cells. Researchers suggest that it may be possible to collect these cells from a patient, genetically modify them, allow them to multiply and then inject them back into the blood stream. The hope is that these cells will then migrate into muscle cells and repair damaged areas. Immune responses would also be kept to a minimum because the patient’s own cells can be used. The initial research has been conducted in a limb-girdle muscular dystrophy mouse model. The mesoangioblasts were isolated from the young mice, modified using a viral vector and then introduced to an artery in the mice. Results showed that these cells were able to produce new muscle cells and that delivery by the blood vessels targeted multiple muscles. While the initial study used limb-girdle mouse models, the research remains important for Duchenne patients as similar rules could be applied to treat this disease.

An interesting study published in June 2003 by Speer et al (22) investigated how muscle stem cells grow and multiply in Duchenne patients. Muscle cell proliferation (multiplication of cells) is decreased in Duchenne muscular dystrophy patients. This may be due to an increase in muscle cell proteins known as p21 and p57 in muscle cells. The study attempted to use oligonucleutides (as mentioned before) to change the DNA in muscle cells and decrease the amount of p21 and p57 protein that were produced. It was hoped that this would then increase muscle cell proliferation in muscles. The investigation had some positive effects increasing cell proliferation. However it was found that some of the oligonucleotides had a toxic effect and caused death in many of the cells. The investigation introduced a possible new method of using stem cells, but research is in its preliminary stages and more study is needed.


Transfer of myoblasts

Encouraging results were seen in a study by Jacques Tremblay in Canada (23) earlier this year when three boys with Duchenne muscular dystrophy received muscle derived stem cells from a close relative and began producing dystrophin protein in a small number of fibres. The boys did not reject the stem cells as a powerful immunosuppressant was given. In the experiment 25 injections were given in one cm3 of muscle. Four weeks later the muscle biopsies showed that one boy was producing dystrophin protein in 9% of the fibres, one boy was producing 6.8% and the other was producing 11%. Although the results are relatively good, it was a very small area that was treated and a functional improvement was not expected. A more effective delivery technique would need to be perfected to target all muscle.

Although many of these experiments have seen interesting results they are often met with similar hurdles. Immune rejection is a common problem. The use of the patient’s own adult cells may alleviate this problem but reactions still may occur if viral vectors are used or when new dystrophin is produced. Finding an efficient way of delivering cells to the body is also being investigated, past experiments have shown that straight injection of stem cells into muscle has only a localised effect due to limited movement and death of cells. Some new techniques have been developed here such as using stem cells located in the vascular system but more research is necessary if this is to become an effective treatment. Much of the work is still at animal model level and while animals such as the mdx mouse are excellent models to work with, these results ultimately need to be checked in humans.


Utrophin

Utrophin is a protein which has a similar structure to dystrophin. In animal model studies it has been able to functionally replace the dystrophin protein and thus may present a therapeutic option for Duchenne. During foetal development utrophin can be found in much higher concentrations within muscle cells where we normally see dystrophin. However after birth this position shifts to the neuromuscular junction. It has been noted that in Duchenne patients and female carriers, levels of utrophin protein expression is increased. It is presumed that this is a compensatory mechanism for the absence or reduced levels of dystrophin protein. By substantially increasing levels of utrophin protein (called upregulation) in young dystrophic mdx mice, the pathology of muscular dystrophy was prevented.

There are some advantages in using utrophin as a therapy for Duchenne muscular dystrophy. Firstly as it is naturally produced in the body and in patients with Duchenne muscular dystrophy, this would avoid potential immune responses to the introduction of dystrophin protein. Secondly if upregulation could be controlled using a drug, it would be possible to express utrophin in all muscle, thus over coming the problem of finding an efficient delivery system. Some drugs have been identified that can upregulate utrophin but so far this has not been to therapeutic levels.

A very recent study in Kansas, USA published in June 2004 (24) has investigated the possibility of Okadaic Acid as promoter for utrophin production. The acid could be used to control the utrophin promoter thus increasing expression of the protein and ameliorate the symptoms of the condition. So far the studies have been carried out using mouse model cells and the effects on human cells have not been determined.


Pharmacological approaches

Pharmacological approaches refer to the use of drugs as possible therapies for Duchenne muscular dystrophy. Part of these studies include investigation of drugs already registered and marketed for use for other conditions. The advantage of testing known drugs is that if one was found to be helpful for Duchenne muscular dystrophy it could easily be fast tracked through clinical trials as it is already registered. Examples of drugs include steroids, growth factors, antibiotics, calcium inhibitors and anti-inflammatories. There has recently been some interest in the use of growth factors to increase muscle bulk and also certain chemicals which appear to have the ability to support the complex of proteins (which normally includes dystrophin) found in the muscle cell membrane and therefore alleviate some of the symptoms associated with Duchenne muscular dystrophy.

It has been shown that the use of Insulin-like growth factors (IGF) have the ability to increase muscle mass. Research published by Professor Lee Sweeney this year (25) has demonstrated that IGF-1 when injected into the legs of rats and mice and increased muscle growth. These results are interesting as it may have the potential to slow muscle cell breakdown, therefore keeping people with muscular dystrophy active for longer. This would not however provide a cure for the disease as the muscle would still be affected by the same mutation and the dystrophin gene is not replaced or corrected in anyway. This means that the muscle would still eventually breakdown. More research is needed to see if this has any potential for use in humans.

Scientists have shown that myostatin, a gene that regulates muscle mass, may be manipulated to increase muscle mass in dystrophic mice. Myostatin belongs to a family of growth factors and is known as a negative regulator of muscle mass as it acts to prevent muscle growth. A study in the USA published in January 2003 (26) showed that by blocking the action of myostatin using an antibody, the mice produced larger muscles and their grip was improved. This offers hope that it might be used therapeutically for Duchenne muscular dystropy although human studies will need to be performed before its potential is known. Other studies into myostatin such as the one in New Zealand published in September 2003 (27) have also shown that it has a significant effect on satellite cell activity. Satellite cells are present on muscle cells and become active when muscle fibres are damaged in order to make new muscle cells to repair the damage. It is possible that blocking myostatin may activate these satellite cells to produce muscle cells. Further research is needed to determine the effect of activating these stem cells to ensure that their supply is not exhausted.

More recently a boy in Germany (28) with unusually large muscles was identified and genetic tests showed that this was due to a mutation in the myostatin gene. This meant that the boy did not produce myostatin and apart from his very large muscles it does not so far appear to have affected his health, although this will be studied as he grows up. This demonstrates that the effect seen in mice also exists in humans and gives further proof that it might be used therapeutically. It is likely that carefully controlled human trails using myostatin will be run in the foreseeable future. Such studies do not provide a cure, as the mutation would still be present in the muscles, but it may slow muscle degeneration or provide useful knowledge for other areas of research such as stem cell studies.



Scientists in the USA (29) published a paper in October 2003 in which they claimed that an enzyme inhibitor known as MG-132 could rescue the expression of cell membrane proteins. The dystrophin protein is localised at the cell membrane and is associated with a number of other membrane proteins. This network or complex is greatly reduced in patients with Duchenne muscular dystrophy.

This is due, in part, to the action of particular enzymes. Enzymes are natural molecules that are able to breakdown proteins (such as the type that aid digestion). The report suggested that the breakdown of the complex could be prevented by using an enzyme inhibitor (MG-132). The experiments were carried out in mdx mice. It was shown that treatment of the mice with the inhibitor reduced muscle membrane damage and ameliorated the signs of Duchenne muscular dystrophy seen in muscle biopsies. While the study has so far only been conducted in mice it does show the clinical implications for the use of such pharmacological treatments for Duchenne muscular dystrophy.


The use of steroids in Duchenne muscular dystrophy patients is now common practice as a means of delaying muscle degeneration and prolonging walking. In general, steroids are used in ambulant boys anytime from four years of age. The decision to begin the use of steroids needs to be carefully discussed with families and a fact sheet exists on this subject (there is a Q and A paper on steroids, please contact The Advice and Information Department for details - email info@muscular-dystrophy.org). At present the regime used varies between centres with some boys on a regular daily dose and others taking it for a certain number of days per month. The choice of regime is often determined by the side effects, which are variable between boys. It is hoped that multicentre trials in the UK will begin to determine which regime is most helpful.

Studies such as the one carried out in Italy and published in February 2003 (30) suggested that low doses of Prednisone (equivalent of Prednisolone in the UK) should be started as soon as a definite diagnosis is achieved. The study used boys aged two to four-years-old and gave one group low doses of Prednisone. It was shown after 55 months that this group was still able to get up from the floor whereas two out of three in the control group had lost this ability. The study suggested that steroids are not sufficient for recovering function but can prolong it and therefore should be started early. The study did also highlight the side effects that are normally associated with the use of steroids such as weight gain, and decline in growth rate.

Another side effect of steroids is an increase in the rate of osteoporosis or the weakening of bones, which may increase the risk of fractures.
How steroids exert their effect on muscle cells is not known. The action involves anti-inflammatory properties but it is possible that they increase or reduce gene activities too. If more research is conducted into these areas it may be possible to produce targeted treatments which have less side effects.

Another area of possible therapy is the use of supplements such as creatine in an attempt to build up lean muscle mass. Previous studies have shown mixed results, however a recent study published in May 2004 by scientists in Canada (31) have shown that supplements of Creatine monohydrate can have positive effects in preventing bone degeneration. Other functional tasks such as climbing stairs and activities of daily living did not improve.

What does all this research mean?

Research in 2003 and 2004 has seen many advances into a wide variety of potential therapies. Some of the most exciting research news has come from ‘exon skipping’ and the consortium bid to the Government for funding for this project. This means a positive step forward towards the start of a UK clinical safety trial and a potential treatment for Duchenne muscular dystrophy. Much of the research still remains at the laboratory stage but the hope is that 2004 will bring further advances towards a treatment for Duchenne muscular dystrophy.

References

(1) Yue et al, Microdystrophin gene therapy of cardiomyopathy restores dystrophin-glycoprotein complex and improves sacrolemma integrity in the mdx mouse heart. Circulation 108 (13) 1626-32 (2003)
(2) Takeda S, Gene therapy on muscular dystrophy. No To Hattatsu 36 (2) 117-23 (2004)
(3) Wells DJ et al, High-efficiency plasmid gene transfer into dystrophic muscle. Gene Ther 10 (6) 504-12 (2003)
(4) Murakami T et al, Full-length dystrophin cDNA transfer into skeletal muscle of adult mdx mice by electroporation. Muscle Nerve 27 (2) 237-41 (2003)
(5) Transgene plasmid gene transfer study in Duchenne or Becker muscular dystrophy: cautious first step yields an encouraging outcome. (2003) Research Update (contact Information office)
(6) Liang KW et al, Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA. Gene Ther 11 (11) 901-8 (2004)
(7) Wells DJ et al, Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins. Gene Ther 11(11):884-93 (2004)
(8) Van Deutekom et al, Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am j Human Genet 74 (1) 83-92 (2003)
(9) Wells DJ et al, Enhanced in vivo delivery of antisense oligonucleotides to restore dystrophin expression in adult mdx mouse muscle. FEBS Lett 552 (2-3) 145-9 (2003)
(10) Partridge et al, Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nature Medicine, Volume 9 Number 7. (2003)
(11) Rando TA et al, Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping. Hum Mol Genet 15;12 (10) 1087-99 (2003)
(12) Matsuo M et al, Chimeric RNA and 2’-O, 4’-C-ethylene-bridged nucleic acid have stronger activity than phosophorothioate oligodeoxynucleotides in induction of exon 19 skipping in dystrophin mRNA. Oligonucleotides 14 (1):33-40 (2004)
(13) Sweeny et al. Aminoglycoside antibiotics restore dystrophin function to sketetal muscles of mdx mice. Journal Clin Invest 104 (4): 375-81 (1999)
(14) Comi LI et al, Gentamicin administration in Duchenne patients with premature stop codon. Preliminary results. Acta Myol. 22 (1) 15-21 (20030
(15) Dunant P et al. Gentamicin fails to increase dystrophin expression in dystrophin-deficient muscle. Muscle Nerve 27 (5):624-7 (2003)
(16) Matsuda R et al, Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice. J Biochem 134 (5) 751-8
(17) http://www.ptcbio.com/big/discovery1flash.html
(18) Wodnar-Filipowicz A, Plasticity of stem cells: a change of paradigm? Ther Umsch 59 (11) 565-70 (2002)
(19) Torrente Y et al, Identification of a ourative pathway for the muscle homing of stem cells in a muscular dystrophy model. J Cell Bio 162 (3) 511-20 (2003)
(20) Kunkel LM et al, Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells. Proc Natl Acad Sci USA 101 (10) 3581-6 (2004)
(21) Cossu G et al, Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 301(5632):487-92 (2003)
(22) Speer et al. Transfection of normal primary human skeletal myoblasts with p21 and p57 antisense oligonucleotides to improve their proliferation: a first step towards an alternative molecular therapy approach of Duchenne muscular dystrophy. J mol Med 81 (6) 355-62 (2003)
(23) MDI News Update, Issue16, March 2004.
(24) Werle MJ et al, Okadaic acid augments utrophin in myogenic cells. Neurosci Lett 363 (2) 163-7 (2004)